Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 28 October 2020; Experimental completion date: 11 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

In the range finding test, activated sewage sludge micro organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L (three replicates for the test concentration of 1000 mg/L). The test item was dispersed directly in water (test water used was deinonized reverse osmosis water containing less than 1 mg/L Total Organic Carbon).
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and sealed with parafilm prior to being subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (three replicates at 1000 mg/L).
The pH of the test item dispersions was measured after stirring using a Hach HQ30d Flexi handheld meter and adjusted to between pH 7.0 and 8.0.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro organisms was obtained on 10 November 2020 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK which treats predominantly domestic sewage.

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC overnight prior to use in the test. On the day of collection, determination of the suspended solids level of the activated sewage sludge was carried out. A sample (100 mL) of the activated sewage sludge was filtered by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed three times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.3 g/L and was adjusted to
3.0 g/L prior to the activated sewage sludge (6.5 L) being fed an aliquot of synthetic sewage (325 mL).

The pH of the sample on the day of the test was 7.5 measured using a Hach HQ30d Flexi handheld meter. The suspended solids level of the activated sewage sludge was checked prior to use to ensure the suspended solids concentration was equal to 3.0 g/L.

Synthetic Sewage:
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Each constituent was dissolved in a final volume of 1 L of water with the aid of ultrasonication.
The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in the range finding test was pH 7.2. The pH values were measured using a Hach HQ30d Flexi handheld meter.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20°C

pH:

pH of test preparations ranged from 7.1 to 7.8 after adjustment.

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

Preparation of Test System:
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air at a rate of 0.5 to 1.0 L per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item. The test item vessels were then prepared as described in 'details on test solutions' section. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of approximately 20°C.

Assessments:
Observations:
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.

pH measurements:
The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3 Hour incubation period using a Hach HQ40d Flexi handheld meter.

Oxygen concentration:
The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Measurement of the Respiration Rates:
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol (at concentrations of 3.2, 10 and 32 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Range-Finding Test:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L; however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Based on these results it was considered not necessary to perform a definitive test.

Validation Criteria:
• The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 6.1 mg/L.
• The specific respiration rate of the controls was 31.92 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 4.7%.
All validation criteria for the study were therefore satisfied.

Results with reference substance (positive control):

The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 6.1 mg/L.

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	1	3.9	2	2.2	51.00	-
	2	3.9	3	1.6	46.00	-
	3	5.4	4	2.3	46.50	-
	4	5.0	4	1.8	48.00	-
Test Item	10	4.5	3	2.0	50.00	[4]
	100	4.4	3	1.9	50.00	[4]
	1000 R1	4.3	3	2.3	40.00	16
	1000 R2	4.9	5	1.7	38.40	20
	1000 R3	5.0	5	1.7	39.60	17
3,5-dichlorophenol	3.2	5.8	8	1.8	30.00	37
	10	6.8	10	3.4	20.40	57
	32	8.0	9	7.0	6.67	86

 

-        = Not applicable

R    = Replicate

[ ]  = Increase in respiration rate as compared to controls

 

Inhibition of Respiration Rate

The following results were derived:

	MPP		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	>100	-	-	-
EC20	>1000	-	1.4	-
EC50	>1000	-	6.1	*

It was not possible to calculate an EC₁₀ value for the reference item as the data generated was not suitable for the calculation method used to calculate this value.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O₂/L and 2 mg O₂/L respectively).  This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

 

 -       =    Not applicable

* It was not possible to calculate 95% confidence limits for the reference item due to insufficient number of replicates vessels to enable this calculation.

 

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro organisms gave a 3 Hour EC50 value of greater than 1000 mg/L.

Description of key information

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge.  The method followed was designed to be compatible with the OECD Guidelines for the Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5‑dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3‑Hour EC₅₀ value of greater than 1000 mg/L. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item, 3,5‑dichlorophenol, gave a 3‑Hour EC₅₀ value of 6.1 mg/L thereby confirming the acceptability of the inoculum used in the test system.

 

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L